Pinoresinol stimulates keratinocyte proliferation and downregulates TNF‐α secretion in peripheral blood mononuclear cells: An experimental in vitro study

Abstract Background and Aims Natural coniferous resins are used in traditional medicine for the treatment of skin wounds. Coniferous wood resins (“callus” resin) are a mixture of abietic (resin) acids, lignans such as pinoresinol, and p‐coumaric acid. The wound‐healing properties of resins are thought to be related to their antimicrobial properties, but also to their effects on cell proliferation and inflammation. The purpose of this study was to identify and investigate the effects of novel aqueous dispersions of resin and its main components in the proliferation of human primary keratinocytes in vitro and in the expression of proinflammatory cytokines in human peripheral blood mononuclear cells. Methods The proliferation studies were performed under low and high calcium conditions with or without added growth stimulators at the time points of 2 and 6 days using AlamarBlue Cell Viability Reagent. The cytokine release assay was carried out by incubating the cells with the test articles for 18 h, after which the levels of tumor necrosis factor‐α (TNF‐α), interleukin‐1β (IL‐1β), IL‐6, and IL‐8 were measured in the supernatant by enzyme‐linked immunosorbent assay. Results Resin and the purified lignan PINO, but not p‐coumaric acid or abietic acid (industrial tall oil rosin), enhanced the proliferation of human keratinocytes in vitro and inhibited the expression of TNF‐α, and to a lesser extent the expression of IL‐1β in peripheral blood mononuclear cells. Conclusions In this study, novel aqueous dispersions of spruce resin were used to investigate the effects of main resin components on keratinocyte proliferation and on the expression of key proinflammatory cytokines known to be associated with chronic wounds. The observations suggest that lignans, such as PINO, but not resin acids, are the components of resins that mediate the proliferative and TNF‐α‐suppressing effects. Lignans including PINO were identified as novel potential compounds in the treatment of chronic skin ulcers.


| INTRODUCTION
Natural coniferous "callus" resins collected from injured Norway spruce trees (Picea abies (Linnaeus, 1753; Karsten, 1881) have been used in traditional medicine, particularly in Northern Europe, as a self-remedy in various skin diseases, particularly skin wounds. Wood resins are complex mixtures of several compounds. Of wood resins, the wood oleoresins and rosins extracted industrially from coniferous tall oil are composed exclusively of terpenoid resin acids, mainly abietic acid. The "mature" wood resins from injured trunks of coniferous trees ("callus resin") contain, in addition to resin acids, lignans and p-coumaric acids (PCAs), 1,2 which are secreted slowly from wood cells in the tree bark and mixed with the oleoresin.
Formulated as salves or ointments, wood resins and tall oil rosin are antimicrobial against a wide range of bacteria, fungi, and yeasts. [2][3][4] In human clinical trials, salves prepared from wood "callus" resins also improve the healing of skin ulcers, [5][6][7] indicating that their ingredients enhance skin renewal, possibly by an unspecific mechanism involving the expression of cytokines and growth factors in skin tissues.
In wood resins, lignans and coumaric acid are watersoluble, whereas the terpenoid rosins are, in general, poorly soluble in water, 8 making their investigations difficult in in vitro tests. Novel methodologies allow, however, the preparation of stable aqueous dispersions or emulsions of natural wood rosins. These aqueous solutions contain, in addition to lignans and coumaric acid, small amounts of water-soluble oxidized resin acids. 2 These aqueous dispersions allow reliable in vitro laboratory testing not amenable to salves or ointments. In earlier studies, the novel water dispersions of natural wood resins were shown to be as antimicrobial as salves or ointments, suggesting that the resin acids in both wood resins and tall oil rosins are the components responsible for the antiseptic property of the resins in general. 2 Pinoresinol (PINO), among other lignans, belongs to phytoestrogens that occur naturally in plants and are structurally similar to mammalian estrogens. Although scientific interest in lignans has increased significantly in recent years, very little is known about their possible pharmacological properties in humans. However, it has recently been reported that PINO promotes keratinocyte re-epithelialization 1 and osteoblast proliferation 9 in an in vitro wound healing assay.
The purpose of this study was to identify the resin substances that might play a role in wound healing. In this investigation, we evaluated how the aqueous dispersions of coniferous wood resins (RE) of natural "callus" origin and industrial rosin (RO) originating from tall oil influence the proliferation of skin keratinocytes in vitro and whether they influence the expression of cytokines in human blood cells. The test articles were water dispersions of RE and RO, with rapeseed oil (RY) as a cosolvent. In addition, the tests were performed with water solutions of purified PINO, secoisolariciresinol (SECO), and nortrachelogenin (NTG). PINO and SECO are among the most abundant lignans in RE, whereas NTG is a common lignan found in knots (branch bases), but to a lesser extent in resins of P. abies. 10 The test articles also included purified PCA, an abundant component in RE, but absent in RO.

| Test articles: Aqueous dispersions of rosins, lignans, and PCA
RE is an aqueous dispersion prepared from "callus" resin. Shortly, callus resin collected from trunks of Norway spruce (P. abies) was dissolved in RY, glycerol (GLY), or 1,3-propanediol (DI) and transferred further to saline to reach a stable oil-in-water emulsion. 2 RO was prepared similarly by using the tall oil rosin instead of the "callus" resin. These dispersions were used at various dilutions in the experiments. Identical solvent controls, prepared without RE or RO, were used as controls.
The composition of RE and RO dispersions was analyzed using short-column gas chromatography-flame ionization detection and gas chromatography/mass spectrometry (GC-MS). Component identification was based on retention times and mass spectra (GC-MS) and the quantification was made on short-column GC with betulinol as standard as previously described. 2 The RE dispersion was a mixture of oxidized resin acids, lignans,

| Cell viability assays
Cell viability assays were conducted to assess the effect of test articles in the vehicles at two different concentrations, 10 and 1.

| Keratinocyte proliferation assays
Human keratinocytes were isolated from the foreskins of one healthy infant donor undergoing circumcision. 11 Keratinocytes were cultured with a standard method using a medium containing Keratinocyte-SFM™ (KSFM) (Life Technologies), epidermal growth factor (EGF) (5 ng/ml), bovine pituitary extract (BPE) (50 μg/ml), penicillin (100 U/ml), and streptomycin (100 μg/ml). The cells were seeded into flasks at a density of about 10,000-20,000 cells/cm 2 . The flasks were incubated in 5% CO 2 at 37°C. The medium was changed every 2-3 days. After the primary culture, cells were passaged every 3-6 days and used between passages 3 and 8.

| Cytokine release assay
Human whole blood was obtained from two healthy human subjects (males aged 45 years and females aged 42 years) using heparincoated vacutainers. Peripheral blood mononuclear cells (PBMCs) were immediately isolated from whole blood using a standard density separation protocol (Histopaque 1077; Sigma). Cell viability was assessed by Trypan Blue exclusion and was found to be >99 before transfer to plates. PBMCs were seeded in wells of a 12-well cell culture plate at a density of 500,000 cells/ml in RPMI-1640 medium supplemented with 10 FCS. Cells were allowed to equilibrate in a humidified cell culture (37°C, 5% CO 2 ) incubator for 30 min before the addition of the test articles.
The test articles were added to the wells at the final concentration of 4 or 1. The vehicle (RY) was used to equalize the volume of test article/vehicle added to each well. Beclomethasone, a well-characterized glucocorticoid, was used as a reference compound. Glucocorticoids are corticosteroids that upregulate the expression of anti-inflammatory proteins in the nucleus and repress the expression of proinflammatory proteins. 13 Beclomethasone was prepared as a concentrated stock solution and was diluted in RPMI medium to a final concentration of   interleukin-1β (IL-1β), interleukin-6 (IL-6), and interleukin-8 (IL-8).

| Group definition
The cell viability assays with keratinocytes were performed with 1 and only to screen the test articles for further experiments. In the following keratinocyte proliferation assays, the test articles were tested in high and low calcium conditions, with or without added growth stimulators, for 2 or 6 days. The concentrations used were the same as in the initial screening. The cytokine release assays were performed using the same set of test articles as keratinocyte proliferation assays, supplemented with commercially available Resolain™ scalp tonic, which was used as a control solution for RE. The different test groups and testing conditions are summarized in Table 2.

| Statistical analyses
In tests on cell viability and in proliferation assays, mean (±SD) values of replicates from parallel wells were calculated and used for statistical analysis. Three biological replicates were used in the cell viability assay and five biological replicates were used in proliferation assays.  (Figure 1).  Table 3). Abbreviations: NTG, nortrachelogenin were prepared the same way as RE; PBMC, peripheral blood mononuclear cell; PCA, p-coumaric acid; PINO, pinoresinol; RE, dissolved callus resin dispersed in rapeseed oil and saline; RO, rosin; RY, rapeseed oil dispersed in saline; SECO, secoisolariciresinol.

| Cytokine release from PBMCs
The release, and levels of IL-6 in wells with medium only were essentially zero ( Figure 3C).

F I G U R E 1 Effect of test article and cosolvents on cell viability in keratinocytes in 24-h incubation in concentrations 1 or 10.
Values represent means of three replicates ± SD. Statistical analysis between data sets was carried out by one-way ANOVA followed by Dunnett's posttest. *p < 0.05 and ***p < 0.001 compared to media-only or to corresponding vehicle control. ANOVA, analysis of variance; PBS, phosphate-buffered saline; RE, dissolved callus resin dispersed in rapeseed oil and saline; RY, rapeseed oil dispersed in saline.  shown as mean ± SD from triplicate wells. The "no-LPS" wells represent the basal release of cytokine from unstimulated cells. Statistical analysis between data sets was carried out by one-way ANOVA followed by Dunnett's posttest. ***p < 0.001 and * p < 0.05 denotes significant cytokine release reduction compared to control. ## p < 0.01 and # p < 0.05 denotes a significant elevation of cytokine release compared to the control. ANOVA, analysis of variance; TNF-α, tumor necrosis factor-α; IL-1β, interleukin-1β; LPS, lipopolysaccharide; PBMC, peripheral blood mononuclear cell.
not seen with the RO emulsion containing oxidized resin acids only, suggesting that the lignans are the likely ingredients enhancing keratinocyte proliferation (see Supporting Information: Table 3). This is further supported by the observation that the purified PINO, one of the main lignans in "callus" wood rosins, enhanced keratinocyte proliferation, similar to the RE substance.
Resin acids are the main ingredients in natural coniferous wood rosins in general, in both "callus" origin and industrial extracts of the coniferous tall oil. 1 In aqueous RE or RO dispersions, resin acids occur at concentrations of 150 ppm at maximum and appear as oxidized forms of dehydroabietic acid (see Table 1). According to the present observations, these resin acids do not stimulate keratinocytes to proliferate. This is the case with PCA as well. On the other hand, even though not being keratinocyte stimulators, neither resin acids nor PCA caused observable cell death. Even in experiments lasting up to 6 days, the keratinocyte growth in culture with RO and PCA was equal to those with control cultures.

| Expression of proinflammatory cytokines in PBMCs
Wound healing can be divided into four phases: hemostasis phase,  19 In high glucose conditions, TNF-α has been shown to upregulate TIMP1 expression in keratinocytes resulting in impaired keratinocyte migration, and the targeting of TNF-α has been suggested as a potential therapy to improve diabetic wound healing. 20 Elevated levels of IL-1β have a similar effect to that of TNF-α.
They have been shown to maintain each other's expression and therefore amplify this signal. 21

CONFLICT OF INTEREST
The cytokine release assays were sponsored by, and the test articles were provided by, Repolar Pharmaceuticals Ltd. The research may lead to the development of new products, in which the authors have no business and/or financial interest. The stated financial relationships had no involvement in the study design or in the decision to submit the report for publication.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.

ETHICS STATEMENT
This work has been conducted in accordance with Wiley's Best Practice Guidelines on Publishing Ethics and it has been performed in an ethical and responsible way, with no research misconduct, which includes, but is not limited to data fabrication and falsification, plagiarism, image manipulation, unethical research, biased reporting, authorship abuse, redundant or duplicate publication, and undeclared conflicts of interest.

TRANSPARENCY STATEMENT
The lead author Elias Haapakorva affirms that this manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted; and that any discrepancies from the study as planned (and, if relevant, registered) have been explained.